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1.  Staphylococcus aureus Extracellular Vesicles Carry Biologically Active β-Lactamase 
Gram-positive bacteria naturally produce extracellular vesicles. However, little is known regarding the functions of Gram-positive bacterial extracellular vesicles, especially in the bacterial community. Here, we investigated the role of Staphylococcus aureus extracellular vesicles in interbacterial communication to cope with antibiotic stress. We found that S. aureus liberated BlaZ, a β-lactamase protein, via extracellular vesicles. These extracellular vesicles enabled other ampicillin-susceptible Gram-negative and Gram-positive bacteria to survive in the presence of ampicillin. However, S. aureus extracellular vesicles did not mediate the survival of tetracycline-, chloramphenicol-, or kanamycin-susceptible bacteria. Moreover, S. aureus extracellular vesicles did not contain the blaZ gene. In addition, the heat-treated S. aureus extracellular vesicles did not mediate the survival of ampicillin-susceptible bacteria. The β-lactamase activities of S. aureus soluble and extracellular vesicle-associated BlaZ were similar, but only the extracellular vesicle-associated BlaZ was resistant to protease digestion, which suggests that the enzymatic activity of BlaZ in extracellular vesicles is largely protected by the vesicle structure. Our observations provide evidence of the important role of S. aureus extracellular vesicles in antibiotic resistance, which allows the polymicrobial community to continue to evolve and prosper against antibiotics.
PMCID: PMC3716153  PMID: 23529736
2.  EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles 
Journal of Extracellular Vesicles  2013;2:10.3402/jev.v2i0.20384.
Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria) to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids) present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia ( might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.
PMCID: PMC3760654  PMID: 24009897
nanocosmos; communicasomes; exosomes; microvesicles; outer membrane vesicles; membrane vesicles; web portals; phylogenetic analyses
3.  Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells 
Journal of Extracellular Vesicles  2012;1:10.3402/jev.v1i0.18704.
Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.
PMCID: PMC3760640  PMID: 24009881
colorectal cancer; microvesicles; exosomes; ectosomes; metastasis; biomarker; secretome; APEX; label-free quantitative proteomics; nanoparticle tracking analysis
4.  Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles 
Journal of Extracellular Vesicles  2014;3:10.3402/jev.v3.26913.
Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.
PMCID: PMC4275645  PMID: 25536934
extracellular vesicles; microvesicles; microparticles; exosomes; ectosomes; extracellular RNA
5.  MicroRNA in exosomes isolated directly from the liver circulation in patients with metastatic uveal melanoma 
BMC Cancer  2014;14:962.
Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases. Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells. This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates. The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells.
Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP. Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR. Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls.
The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures. Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls.
Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation. The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-962) contains supplementary material, which is available to authorized users.
PMCID: PMC4320618  PMID: 25510783
Exosomes; Extracellular vesicles; Biomarker; microRNA; Uveal melanoma; Liver perfusion
6.  Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways 
PLoS ONE  2014;9(12):e115170.
Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference–mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.
PMCID: PMC4264882  PMID: 25502753
7.  Extracellular vesicles as emerging intercellular communicasomes 
BMB Reports  2014;47(10):531-539.
All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions. [BMB Reports 2014; 47(10): 531-539]
PMCID: PMC4261509  PMID: 25104400
Cancer; Component; Diagnosis; Exosome; Function; Microvesicle; Therapy; Tumor
8.  Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum 
Journal of Extracellular Vesicles  2014;3:10.3402/jev.v3.24783.
Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.
PMCID: PMC4185091  PMID: 25317276
Depletion protocol; Exosomes; Extracellular vesicles; Fetal bovine serum; RNA
9.  An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus 
PLoS ONE  2014;9(7):e100499.
Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). Staphylococcus aureus secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated α-hemolysin derived from S. aureus in AD pathogenesis. α-hemolysin production from S. aureus was detected using western blot analyses. The cytotoxic activity of α-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated α-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The in vivo effects of α-hemolysin were evaluated by application of soluble and EV-associated α-hemolysin on the mouse skin. The present study showed that increased α-hemolysin was produced by S. aureus colonized on AD patients compared to healthy subjects. α-hemolysin production was also related to AD severity. In addition, EV-associated α-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble α-hemolysin, and α-hemolysin-negative EVs did not induce keratinocyte death. EV-associated α-hemolysin induced necrosis, but soluble α-hemolysin induced apoptosis of keratinocytes. In vivo, skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated α-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated α-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by α-hemolysin-negative EVs. Taken together, α-Hemolysin secreted from S. aureus, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated α-hemolysin is a novel diagnostic and therapeutic target for the control of AD.
PMCID: PMC4084635  PMID: 24992681
10.  Epstein-Barr Virus-Encoded MicroRNA BART15-3p Promotes Cell Apoptosis Partially by Targeting BRUCE 
Journal of Virology  2013;87(14):8135-8144.
Epstein-Barr Virus (EBV) generates a variety of viral microRNAs (miRNAs) by processing the BHRF1 and BamHI A rightward (BART) transcripts. BART miRNAs are expressed in all cells latently infected with EBV, but the functions of most BART miRNAs remain unknown. The results of a cell proliferation assay revealed that miR-BART15-3p inhibited cell proliferation. Fluorescence-activated cell sorting following staining with annexin V or propidium iodide showed that miR-BART15-3p promoted apoptosis. Furthermore, the inhibitor for miR-BART15-3p increased cell growth and reduced apoptosis in EBV-infected cells. Using bioinformatic analyses, we predicted that miR-BART15-3p may target the antiapoptotic B-cell lymphoma 2 (BCL2), BCL2L2, DEAD (Asp-Glu-Ala-Asp) box polypeptide 42 (DDX42), and baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme (BRUCE) mRNAs. The luciferase reporter assay showed that only the 3′ untranslated region (UTR) of BRUCE was affected by miR-BART15-3p. Two putative seed-matched sites for miR-BART15-3p were evident on the BRUCE 3′ UTR. The results of a mutation study indicated that miR-BART15-3p hybridized only with the first seed-matched site on the BRUCE 3′ UTR. miR-BART15-3p downregulated the BRUCE protein in EBV-negative cells, while the inhibitor for miR-BART15-3p upregulated the BRUCE protein in EBV-infected cells without affecting the BRUCE mRNA level. miR-BART15-3p was secreted from EBV-infected gastric carcinoma cells, and the level of miR-BART15-3p was 2- to 16-fold higher in exosomes than in the corresponding cells. Our data suggest that miR-BART15-3p can induce apoptosis partially by inhibiting the translation of the apoptosis inhibitor BRUCE. Further study is warranted to understand the role of miR-BART15-3p in the EBV life cycle.
PMCID: PMC3700184  PMID: 23678170
11.  Cdk5 Phosphorylates Dopamine D2 Receptor and Attenuates Downstream Signaling 
PLoS ONE  2013;8(12):e84482.
The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood, reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3 was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits DRD2 function, providing a novel regulatory mechanism for dopamine signaling.
PMCID: PMC3877277  PMID: 24391960
12.  Extracellular Vesicles Derived from Gut Microbiota, Especially Akkermansia muciniphila, Protect the Progression of Dextran Sulfate Sodium-Induced Colitis 
PLoS ONE  2013;8(10):e76520.
Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammatory bowel disease (IBD). However, owing to the complexity of the gut microbiota, our understanding of the roles of commensal and pathogenic bacteria in the maintenance of immune homeostasis in the gut is evolving only slowly. Here, we evaluated the role of gut microbiota and their secreting extracellular vesicles (EV) in the development of mucosal inflammation in the gut. Experimental IBD model was established by oral application of dextran sulfate sodium (DSS) to C57BL/6 mice. The composition of gut microbiota and bacteria-derived EV in stools was evaluated by metagenome sequencing using bacterial common primer of 16S rDNA. Metagenomics in the IBD mouse model showed that the change in stool EV composition was more drastic, compared to the change of bacterial composition. Oral DSS application decreased the composition of EV from Akkermansia muciniphila and Bacteroides acidifaciens in stools, whereas increased EV from TM7 phylum, especially from species DQ777900_s and AJ400239_s. In vitro pretreatment of A. muciniphila-derived EV ameliorated the production of a pro-inflammatory cytokine IL-6 from colon epithelial cells induced by Escherichia coli EV. Additionally, oral application of A. muciniphila EV also protected DSS-induced IBD phenotypes, such as body weight loss, colon length, and inflammatory cell infiltration of colon wall. Our data provides insight into the role of gut microbiota-derived EV in regulation of intestinal immunity and homeostasis, and A. muciniphila-derived EV have protective effects in the development of DSS-induced colitis.
PMCID: PMC3811976  PMID: 24204633
13.  Three-Dimensional Imaging of Hepatic Sinusoids in Mice Using Synchrotron Radiation Micro-Computed Tomography 
PLoS ONE  2013;8(7):e68600.
Hepatic sinusoid, the smallest vessel in the liver, plays important roles in hepatic microcirculation. Although the structure of the hepatic sinusoids affects diverse functions of the liver, little is known about morphological alterations in the sinusoids under pathological conditions. In this study, we show that the structure of hepatic sinusoids can be identified three-dimensionally in normal and carbon tetrachloride-injured mouse liver, using the absorption mode of synchrotron radiation micro-computed tomography. We observed that the hepatic sinusoidal structure on tomographic slice images was similar to that on histological images of normal and acutely injured mice. Moreover, centrilobular necrosis and structural alterations of the sinusoids in the necrotic region were detectable on tomographic slice and volume-rendered images of the acutely injured mice. Furthermore, quantitative analyses on 3D volume-rendered images of the injured sinusoid revealed decrease in the volume of the sinusoid and connectivity of the sinusoidal network. Our results suggest that the use of synchrotron radiation micro-computed tomography may improve our understanding of the pathogenesis of hepatic diseases by detecting the hepatic sinusoids and their alterations in three-dimensional structures of the damaged liver.
PMCID: PMC3702620  PMID: 23861925
14.  Outer Membrane Vesicles Derived from Escherichia coli Up-Regulate Expression of Endothelial Cell Adhesion Molecules In Vitro and In Vivo 
PLoS ONE  2013;8(3):e59276.
Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1-/- and TLR4-/- mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.
PMCID: PMC3597602  PMID: 23516621
15.  Acetyl salicylic acid inhibits Th17 airway inflammation via blockade of IL-6 and IL-17 positive feedback 
T-helper (Th)17 cell responses are important for the development of neutrophilic inflammatory disease. Recently, we found that acetyl salicylic acid (ASA) inhibited Th17 airway inflammation in an asthma mouse model induced by sensitization with lipopolysaccharide (LPS)-containing allergens. To investigate the mechanism(s) of the inhibitory effect of ASA on the development of Th17 airway inflammation, a neutrophilic asthma mouse model was generated by intranasal sensitization with LPS plus ovalbumin (OVA) and then challenged with OVA alone. Immunologic parameters and airway inflammation were evaluated 6 and 48 h after the last OVA challenge. ASA inhibited the production of interleukin (IL)-17 from lung T cells as well as in vitro Th17 polarization induced by IL-6. Additionally, ASA, but not salicylic acid, suppressed Th17 airway inflammation, which was associated with decreased expression of acetyl-STAT3 (downstream signaling of IL-6) in the lung. Moreover, the production of IL-6 from inflammatory cells, induced by IL-17, was abolished by treatment with ASA, whereas that induced by LPS was not. Altogether, ASA, likely via its acetyl moiety, inhibits Th17 airway inflammation by blockade of IL-6 and IL-17 positive feedback.
PMCID: PMC3584657  PMID: 23306703
Acetyl salicylic acid; IL-6; IL-17A; STAT3; Th17
16.  Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation 
PLoS Biology  2012;10(12):e1001450.
Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.
Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
PMCID: PMC3525526  PMID: 23271954
17.  Vascular Endothelial Growth Factor Is a Key Mediator in the Development of T Cell Priming and Its Polarization to Type 1 and Type 17 T Helper Cells in the Airways 
Chronic inflammatory airway diseases including asthma are characterized by immune dysfunction to inhaled allergens. Our previous studies demonstrated that T cell priming to inhaled allergens requires LPS, which is ubiquitously present in household dust allergens. In this study, we evaluated the role of vascular endothelial growth factor (VEGF) in the development of T cell priming and its polarization to Th1 or Th17 cells when exposed to LPS-contaminated allergens. An asthma mouse model was induced by airway sensitization with LPS-contaminated allergens and then challenged with allergens alone. Therapeutic intervention was performed during allergen sensitization. The present study showed that lung inflammation induced by sensitization with LPS-contaminated allergens was decreased in mice with homozygous disruption of the IL-17 gene; in addition, allergen-specific Th17 immune response was abolished in IL-6 knockout mice. Meanwhile, in vivo production of VEGF was up-regulated by airway exposure of LPS. In addition, airway sensitization of allergen plus recombinant VEGF induced both type 1 and type 17 Th cell (Th1 and Th17) responses. Th1 and Th17 responses induced by airway sensitization with LPS-contaminated allergens were blocked by treatment with a pan-VEGF receptor (VEGFR; VEGFR-1 plus VEGFR-2) inhibitor during sensitization. These effects were accompanied by inhibition of the production of Th1 and Th17 polarizing cytokines, IL-12p70 and IL-6, respectively. These findings indicate that VEGF produced by LPS plays a key role in activation of naive T cells and subsequent polarization to Th1 and Th17 cells.
PMCID: PMC3385973  PMID: 19786548
19.  Therapeutic Effects of Autologous Tumor-Derived Nanovesicles on Melanoma Growth and Metastasis 
PLoS ONE  2012;7(3):e33330.
Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination.
PMCID: PMC3305328  PMID: 22438914
20.  Protective effects of basic fibroblast growth factor in the development of emphysema induced by interferon-γ 
Experimental & Molecular Medicine  2011;43(4):169-178.
Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-γ in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-β, is important in wound healing. We investigated the role of FGF2 in IFN-γ-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-γ-induced emphysema, lung targeted IFN-γ transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 µg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-γ in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-γ but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-γ-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.
PMCID: PMC3085735  PMID: 21297377
asthma; emphysema; fibroblast growth factor 2; interferon-γ; pulmonary eosinophilia
21.  Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses 
Experimental & Molecular Medicine  2010;42(10):721-730.
Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.
PMCID: PMC2966746  PMID: 20841959
asthma; interferon-γ; interleukin-17; neutrophils; nitric oxide synthase type II; RNA viruses; Th1 cells
22.  IL-12-STAT4-IFN-γ axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model 
Experimental & Molecular Medicine  2010;42(8):533-546.
IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-γ-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-γ over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-γ-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-γ expression were impaired in allergen-challenged IL-12Rβ2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Rα-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-γ. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-γ axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
PMCID: PMC2928926  PMID: 20592486
asthma; interferon-γ; interleukin-12; interleukin-13; respiratory hypersensitivity; Th2 cells
23.  A serum-stable branched dimeric anti-VEGF peptide blocks tumor growth via anti-angiogenic activity 
Experimental & Molecular Medicine  2010;42(7):514-523.
Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.
PMCID: PMC2912478  PMID: 20543548
angiogenesis inhibitors; colorectal neoplasms; peptides; receptors, vascular endothelial growth factor; vascular endothelial growth factors
24.  Outer Membrane Vesicles Derived from Escherichia coli Induce Systemic Inflammatory Response Syndrome 
PLoS ONE  2010;5(6):e11334.
Sepsis, characterized by a systemic inflammatory state that is usually related to Gram-negative bacterial infection, is a leading cause of death worldwide. Although the annual incidence of sepsis is still rising, the exact cause of Gram-negative bacteria-associated sepsis is not clear. Outer membrane vesicles (OMVs), constitutively secreted from Gram-negative bacteria, are nano-sized spherical bilayered proteolipids. Using a mouse model, we showed that intraperitoneal injection of OMVs derived from intestinal Escherichia coli induced lethality. Furthermore, OMVs induced host responses which resemble a clinically relevant condition like sepsis that was characterized by piloerection, eye exudates, hypothermia, tachypnea, leukopenia, disseminated intravascular coagulation, dysfunction of the lungs, hypotension, and systemic induction of tumor necrosis factor-α and interleukin-6. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of sepsis, suggesting OMVs as a new therapeutic target to prevent and/or treat severe sepsis caused by Gram-negative bacterial infection.
PMCID: PMC2893157  PMID: 20596524
25.  Aspirin attenuates the anti-inflammatory effects of theophylline via inhibition of cAMP production in mice with non-eosinophilic asthma 
Theophylline is commonly used to treat severe asthma and chronic obstructive pulmonary disease (COPD) characterized by non-eosinophilic inflammation. Acetyl salicylic acid (ASA) is one of the most widely used medications worldwide, but up to 20% of patients with asthma experience aggravated respiratory symptoms after taking ASA. Here we evaluated the adverse effect of ASA on the therapeutic effect of theophylline in mice with non-eosinophilic asthma. A non-eosinophilic asthma mouse model was induced by airway sensitization with lipopolysaccharide-containing allergen and then challenged with allergen alone. Therapeutic intervention was performed during allergen challenge. Theophylline inhibited lung inflammation partly induced by Th1 immune response. ASA attenuated the beneficial effects of theophylline. However, co-administration of the ASA metabolite salicylic acid (SA) showed no attenuating effect on theophylline treatment. The therapeutic effect of theophylline was associated with increase in cAMP levels, which was blocked by co-treatment of theophylline and ASA. ASA co-treatment also attenuated the anti-inflammatory effects of a specific phosphodiesterase 4 inhibitor. These results demonstrate that ASA reverses anti-inflammatory effects of theophylline, and that ASA exerts its adverse effects through the inhibition of cAMP production. Our data suggest that ASA reverses lung inflammation in patients taking theophylline, although clinical evidence will be needed.
PMCID: PMC2811820  PMID: 19887894
adverse effect; aspirin; asthma, aspirin-induced; cyclic AMP; cyclic nucleotide phosphodiesterases, type 4; drug toxicity; pneumonia; theophylline

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